The goal of this study was to investigate whether waggle dancers produce and release into the air chemical compounds that distinguish them from other foragers. We addressed this first goal by using solid phase microextraction (SPME) and gas chromatography coupled with mass spectrometry (GC/MS). If these distinguishing compounds are semiochemicals or pheromones, thenadding these compounds into the hive will affect the behavior of foraging bees. To test whether the compounds are behaviorally active, we measured foraging activity before and after we injected the volatilized compounds into the hive. Our results show that honey bee waggle dancers produce four characteristicvolatile compounds that increase foraging activity.

A chromatogram of an air sample taken from the dance-floor area on the bottom hive frame, where waggle dancers were present (A) and of a sample from the top frame, where honey was stored and no waggle dancers (but other bees) were present (B). Relative amount of fragments is measured in thousands of counts. Peaks 1, 2, 3, and 4 are Z-(9)-tricosene, tricosane, Z-(9)-pentacosene and pentacosane, respectively, and these peaks distinguish the air over dancing bees from the air over nondancing bees.

Relative abundance of Z-(9)-tricosene, tricosane, Z-(9)-pentacosene, and pentacosane sampled from three types of bees. The height of each bar indicates the mean peak area for each compound from GC/MS chromatograms. The relative abundance of compounds is significantly higher for waggle-dancing foragers (black bars) than for either nondancing foragers (hatched bars) or nonforaging bees (white bars, for statistical tests, see text). The relative abundance of compounds for nondancing foragers does not differ from that for nonforaging bees, with the exception of Z-(9)-tricosene. Error bars represent the standard error of the average of six daily mean values, each daily mean comprising a sample of two–three bees of each type. Peak areas represent relative abundance only and thus do not enable comparison among different compounds.

Figure 3

Comparison of Vigorous and Less-Vigorous Dancers

The relative abundance for Z-(9)-tricosene, tricosane, Z-(9)-pentacosene, and pentacosane tended to be higher for vigorous (n = 5, black bars) than less-vigorous (n = 13, white bars) dancers. Vigor of dancing was assessed subjectively by one experimenter and included appearance of abdomen movement and the rate of waggle runs. More vigorous dancers appeared to move their abdomen farther to the side and perform more waggle runs than less-vigorous dancers. Error bars represent the standard error. Statistical tests are given in the text.

Comparison of foraging activity in Colony 2 following injection of the TTP solution versus pure hexane. The normalized mean number of bees exiting the hive following Injection 2 (during minutes 25–32) was greater during TTP trials (black bars) than during Hexane trials (white bars). Results were similar for Colony 1. Statistical tests are given in the text.

We also observed within TTP trials an increase in the number of bees exiting the hive following Injection 2, as compared with the number exiting following Injection 1, but this was significant for only one colony (two-sample T-test; Colony 1: T = −1.63, p = 0.121, df = 18; Colony 2: T = −2.66, p = 0.013, df = 28). This increase could possibly be the result of circadian foraging patterns, but this would not explain the lack of such an effect during Hexane trials (two-sample T-test; Colony 1: T = 0.83, p = 0.415, df = 18; Colony 2: T = −0.68, p = 0.502, df = 24), which were conducted at the same time of day as TTP trials.

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Discussion

We have identified in this study four compounds that are unique to waggle-dancing bees and that are behaviorally active. This waggle-dance scent originates from the waggle dancers themselves; it is not acquired from the environment while foraging, nor is it a byproduct of a bee's age or task, because it is emitted in only minute quantities by nondancing foragers returning from the same food source.

The chemical nature and source of the compounds of the waggle-dance scent differ from those of the bumblebee foraging recruitment pheromone [15]. The main compounds of the bumblebee recruitment pheromone were identified as eucalyptiol, ocimene, and farnesol, which are terpenederivatives. These compounds are produced in the bees' tergalglands. Their different chemistry and source suggest different evolutionaryorigins for the bumblebee foraging recruitment pheromone and the waggle-dance scent of honey bees. Hence, it is unlikely that the waggle-dance scent of honey bees has evolved from the bumblebee foraging recruitment pheromone.

Whereas the waggle-dance signal is likely a signal intended for new recruits, two other groups of bees, namely foragers alreadydevoted to a food source and in-hive receiver bees, could glean cues from the waggle-dance scent. Foragers that are already devoted to a food source do not readily follow new dances if their source becomesunavailable, but ratherwait for it to replenish [8]. Because the waggle-dance scent does not seem to identify specific food sources, it can provide only limited information to these foragers. However, high concentrations of the scent could alert them to generally good foraging conditions. This could be useful at the beginning of daily foraging or if foraging can be resumed after a spell of badweather. This mechanism may have been responsible for the effect observed in an earlier preliminary study, in which the number of foragers visiting an empty feeder increased following exposure to the air from a foraging colony [13]. In our experiments, however, the increase in foragers was not likely caused by already-devoted foragers for three reasons. First, experiments were done well after the time that colonies started daily foraging. Second, external conditions such as the weather were remarkablystable, which made strongfluctuations in the numbers of already-devoted foragers unlikely. Third, we did not record a conspicuous drop in the number of bees that left the hive after the initial increase (Figure 4); if the increase would have been duemostly to already-devoted foragers, we would have expected a quick drop to original levels once these foragers found that there was no change in food-source profitability. However, it is possible that such a drop could be hidden by the more substantial numbers of newly recruited foragers. Receiver bees are the second group of bees that could glean cues from the waggle-dance scent. Receiver bees unload nectar from newly returned foragers, which relieves foragers of the time-consumingsearch for empty storagecells. A high concentration of the waggle-dance scent would indicate a high demand for receiver bees, and could help attract receiver bees to the dance floor. The tremble dance, which is performed by successful foragers that perceive a shortage in receiver bees [22], may help to additionallyspread the scent to potential receiver bees.

We compared the chemical profile of the air over dancing bees to that over nondancing bees, and we also compared the chemical profile of waggle dancers' abdomens to those of both nondancing foragers and nonforaging bees. For air samples, we exposed the fiber for 5 consecutive min approximately 2 cm above the surface of the comb. For abdomen profiles, we briefly touched the fiber to the tip of the abdomen of an individual bee [23–26]. Waggle dancers were sampled during a waggle run, shortly after they began dancing. Sampled bees were observed from the moment they entered the hive until the sample was taken. It is possible that we classified as nondancing foragers bees that danced before observation began (e.g., in the entrance tunnel to the hive) or that danced after SPME sampling, but this would bias our results in only a conservativedirection.

Behavioral tests.

TTP trials consisted of 32 min during which we made two injections: Injection 1, 50 μl of pure hexane, during minute 1; Injection 2, 50 μl of TTP solution, during minute 16. Hexane trials were similar to TTP trials except that Injection 2, like Injection 1, consisted of 50 μl of pure hexane.

The TTP solution contained Z-(9)-tricosene, tricosane, and pentacosane, each diluted 1:100 in hexane and mixed at a ratio of 1:2:3, respectively (this ratio produced chromatograms with peakheights that approximately matched those from samples of waggle dancers), and then further diluted 1:10 in hexane. To volatilize the liquid TTP solution, we injected it into a heatedglasstube with 0.5-cm ID. Immediately after injection, we inserted into the tube a fan to blow the vaporized solution through the tube and into a funnel (10-cm diameter) positioned 1 cm above the comb surface of a colony's dance floor. The identical method was used to volatilize and deliver hexane only during Hexane trials (Injection 1: hexane, Injection 2: hexane). To avoidcontamination between TTP and Hexane trials, we used separateequipment for TTP solution and hexane. The temperature of the gaseous mixture arriving on the dance floor was maintained between 35 °C and 40 °C. At least one waggle dancer was present on the dance floor during each trial.

We conducted each trial on a different day between 14 July and 8 October 2004. To avoid seasonaleffects, we randomized the type of trial (TTP or Hexane) performed each day. To reduce the effect of time of day, trials for a colony started during the same hourevery day. Trials for the first colony started between 1200 and 1300, and for the second colony between 1030 and 1130. To further account for day and time effects, the data for each trial were normalized by dividing each 1-min count by the average number of bees exiting the hive per minute during the 10 min immediately preceding the trial.

Statistics.

To compare the presence of the four compounds on the abdomens of waggle dancers with either nondancing foragers that returned from the same unscented food source or nonforaging bees, we used a one-way ANOVA for each compound using Box-Coxtransformed data, and a Tukey HSD for unequal n, with data for both colonies pooled.

To compare the effect of injection of TTP solution with injection of hexane, we used two-sample T-tests on normally distributed sample groups with equal variances. To account for day and time effects, the data for each trial were normalized by dividing each 1-min count by the average number of bees exiting the hive per minute during the 10 min immediately preceding the trial.

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